P16ink4a derived peptides for prophylaxis and therapy of hpv-associated tumors and other p16ink4a expressing tumors

ABSTRACT

Described are particular fragments of the cyclin-dependent kinase inhibitor p16 capable of increasing IFN-γ secretion of T cells or inducing proliferation of T cells and the use of said fragments for immunizing an individual against HPV-associated or other p16 INK4a  expressing carcinomas, preferably advanced carcinomas.

This application is a divisional application of U.S. application Ser.No. 13/714,273, filed Dec. 13, 2012. The contents of theabove-identified application is incorporated herein by reference in itsentirety.

REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM

The Sequence Listing is concurrently submitted herewith with thespecification as an ASCII formatted text file via EFS-Web with a filename of Sequence Listing.txt with a creation date of Mar. 27, 2013, anda size of 3.05 kilobytes. The Sequence Listing filed via EFS-Web is partof the specification and is hereby incorporated in its entirety byreference herein.

TECHNICAL FIELD

The present invention relates to particular fragments of thecyclin-dependent kinase inhibitor p16^(INK4a) and the use of saidfragments for immunizing an individual against p16^(INK4a)-expressingtumors.

BACKGROUND OF THE INVENTION

Several million people fall ill with, and die of, carcinomas world-wideevery year. These mortality rates have remained unchanged for many yearsdespite intensive therapy research. Until now, patients suffering fromcarcinomas often have to undergo carcinoma-removing surgery orchemotherapy or radiation therapy. However, this is accompanied by verymassive side-effects which then contribute to the mortality rates ofpatients suffering from carcinomas. Interestingly, humanpapillomaviruses (HPV) are associated with the development of over 5% ofall cancers (Parkin and Bray, 2006). Prophylactic HPV vaccination isalready available but shows no therapeutic effects in already infectedpeople (Hildesheim et al., 2007). Thus, there is a need of noveltreatment options.

SUMMARY OF THE INVENTION

It is the object of the present invention to provide a means for therapyand prophylaxis of HPV-associated tumors and other p16^(INK4a)expressing tumors.

According to the invention this is achieved by the subject mattersdefined in the claims. The studies leading to the present inventionemanated from the observation that the cellular protein p16^(INK4) isexpressed in HPV infected cells when the cells achieve a transformed andpossibly malignant phenotype due to the disruption of a negativefeedback loop by the HPV oncogene product E7 (Sano et al., 1998; Klaeset al., 2001). Thus, p16^(INK4a) is strongly expressed in virtually allHPV-induced carcinomas and high grade pre-neoplasias, includingcervical, vulvar, vaginal, penile, anal and head and neck tumors(Ishikawa et al., 2006; Samama et al., 2006; Missaoui et al., 2006;Santos et al., 2006; Roma et al., 2008; Hafkamp et al., 2003). Underphysiological conditions p16^(INK4) is only expressed in cells thatundergo subsequent senescence and, thus, is barely found expressed innormal tissues (Beauséjour et al., 2007). Besides the HPV-oncogenedriven p16^(INK4a)-expression in HPV-associated tumors, p16^(INK4a) isfound also overexpressed in various tumors not associated with an HPVinfection or in tumors where HPV has been found but a viralcarcinogenesis is not proven, including a fraction of melanoma andnon-melanoma skin cancers (Nindl et al., 2004; Busch et al., 2010), lungcancers (Leversha et al., 2003; Esposito et al., 2004), esophageal,gastric and colorectal cancers (Ding et al., 2010; Kim et al., 2005) andkidney, bladder, ovarian, endometrial and breast cancers (Ikuerowo etal. 2007; Buza et al., 2010; Giordano et al., 2008; Giordano et al.,2007; di Vinci et al., 2005). It is known that mutations of theretinoblastoma tumor suppressor gene result in upregulation ofp16^(INK4a) expression (Okamoto et al., 1994). However, the underlyingmechanisms for the strong p16INK4a expression in these instances aremost likely more heterogenic and not finally understood.

Overexpression of endogenous gene-products in cancer cells has sincelong been recognized as a valuable source for tumor associated antigens.Immune responses against such antigens have been observed in variouscancer patients and have been increased in immunotherapeutic trials(Jäger et al., 2003; Finn, 2008; Rescigno et al., 2007).

During the experiments leading to the present invention it could bedemonstrated that T lymphocytes isolated from peripheral blood samplesof healthy individuals can be specifically stimulated in vitro withp16^(INK4a) derived peptides and that CD4+ and CD8+ T cells fromcervical cancer patients show spontaneous reactivity against the samep16^(INK4a) peptide and give rise to cytotoxic T cell lines that areable to attack and kill co-cultured HLA-matched p16^(INK4a) loaded cellsand cervical cancer cell lines. In other words, fragments of p16^(INK4)are highly immunogenic inducing a very strong immune response againstp16^(INK4). Further, it has been demonstrated that also humoral immuneresponses are detectable against p16^(INK4a).

The described expression pattern of p16^(INK4) and the finding ofspontaneous immune responses against p16^(INK4) that are not associatedwith any autoimmune diseases make p16^(INK4) a promising candidate forimmunization of patients with p16^(INK4a) expressing cancers. Anactively induced strong immune response against p16^(INK4) couldspecifically destroy HPV-transformed cells and otherp16^(INK4a)-expressing cancer cells. Vaccination of donor T cells withthese p16^(INK4) peptides was performed in cell culture experiments.Additional experiments using the p16^(INK4) peptides revealedspontaneous T cell responses in cervical cancer patients confirming thatparticular p16^(INK4) peptides are immunogenic also in vivo. Thus,prophylaxis or therapy of cancer based on immunization using thesepeptides should have several benefits for a patient. p16^(INK4) isstrongly expressed in all HPV-associated cancers irrespective of the HPVtype and in various other cancer types. Severe side effects ofp16^(INK4) immunization are not expected, because p16^(INK4) is barelyexpressed in normal tissues and no autoimmune phenomena have beenobserved in individuals with spontaneous immune responses againstp16^(INK4). Finally, immune evasion due to antigen loss is very unlike,because p16^(INK4) expression is intricately linked to the malignantphenotype of the tumor cell.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows ELISpot (interferon gamma) results before (A) and after (B)stimulation of donor T cells with the p16^(INK4a) peptides.

The spot count is normalized by subtracting background spot detection inwells without peptides. In B the increased spot count in cellsstimulated with the peptides p16INK4a_(—)37-63, p16INK4a_(—)51-80 andp16INK4a_(—)73-104 compared to day 0 becomes clear. CEF=CMV, EBV,influenza (flu) peptide mix positive control.

FIG. 2 shows ELISpot (interferon gamma) results in 23 patients (Tx andFx) and 15 healthy controls (BCx) against the positive control virus mix(CEF) and against the seven 30mer p16^(INK4a) peptides (Table 1).

Results are background adjusted and only spots above the cut-off (2times the spots in the negative control well+2 standard deviations ofreactivity against the respective p16^(INK4a) peptide) are considered.Two individuals had CD4⁺ responses, the remaining had CD8⁺ responses.na=not analyzed.

FIG. 3 shows Chromium release assay.

CD8 T cells from a cervical cancer patient (HLA A2 A3 B7 B15 Cw3 Cw7)induce lysis of HLA-matched B cells loaded with p16INK4a_(—)37-63 (blacksquares) but not of the same B cells without the p16^(INK4a) peptide(open triangles). Cervical cancer cell lines HeLa (p16^(INK4a)+, HLAA68, B15, B95, Cw7 Cw12) and Caski (p16^(INK4a)+, HLA A2, A3, B7, B37,Cw5, Cw7) are lysed while no lysis of ME180 and K562 is detected.

FIG. 4 shows proliferation of peripheral blood mononuclear cells fromwomen with cervical dysplasias after stimulation with the seven p16INK4apeptides (Table 1).

Shown are optical densities of the BrdU proliferation assay performedafter incubation of PBMCs with p16INK4a peptides, positive controls(mitogen PHA and tetanus toxoid) and negative controls (no antigen).Dashed lines indicate cut-off for positive response. Asterisks indicateproliferation inducing p16INK4a peptides. Shown are the results fromthree reacting patients and one negative patient.

FIG. 5 shows erological p16^(INK4a) reactivity in Western blot.

(a) (1) Silver stain of purified His-tagged p16^(INK4a). (2) Westernblot of purified His-tagged p16^(INK4a) with the monoclonal p16^(INK4a)antibody E6H4.

(b) 6 representative positive sera are shown. The protein sizecorresponds to the reaction of the monoclonal antibody (E6H4) againstthe recombinant protein.

(c) Examples of negative sera (1 and 2).

(d) p16^(INK4a) serum reactivity before (1) and after (2) preincubationof serum with recombinant p16^(INK4a). (3) Detection of p16^(INK4a)protein precipitated by serum obtained from a cervical cancer patientand detected by a directly HRP-labeled monoclonal p16^(INK4a) antibody(from (Reuschenbach et al., 2008).

DETAILED DESCRIPTION OF THE INVENTION

The present invention, thus, relates to particular fragments of thecyclin-dependent kinase inhibitor p16^(Ink4) capable of inducing animmune response against p16^(INK4a). An immune response is defined as acondition fulfilling at least one of the following criteria: 1. Theinduction of CD8-positive T cells, as detectable by cytotoxicity assaysor IFN-gamma secretion or perforin expression or granzyme B expressionor other cytokines that may be produced by CD8-positive T cells,measurable as above background by ELISpot or intracellular cytokinestaining or cytokine ELISA or equivalent methods. 2. The induction ofCD4-positive T cells, as detectable by cytokine secretion measurable asabove background by ELISpot or intracellular cytokine staining orcytokine ELISA or equivalent methods. Cytokines may comprise IFN-alpha,IFN-gamma, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, IL-17,TNF-alpha, TGF-beta or other cytokines that may be produced byCD4-positive T cells. 3. The induction of antibodies, as detectable byWestern blot, ELISA and equivalent or related methods. 4. The inductionof any kind of cellular Immune response not mediated by CD8-positive orCD4-positive T cells as described in 1 and 2.

Fragments of p16^(INK4a) are consisting of

(a) any one of the following amino acid sequences:

(SEQ ID NO: 1) (a₁) MEPAAGSSMEPSADWLATAAARGRV; (SEQ ID NO: 2) (a₂)TAAARGRVEEVRALLEAGALPNAPNSY; (SEQ ID NO: 3) (a₃)LPNAPNSYGRRPIQVMMMGSARVAELL; (SEQ ID NO: 4) (a₄)VMMMGSARVAELLLLHGAEPNCADPATLTR; (SEQ ID NO: 5) (a₅)ADPATLTRPVHDAAREGFLDTLVVLHRAGARL; (SEQ ID NO: 6) (a₆)HRAGARLDVRDAWGRLPVDLAEELGHRDVAR; (SEQ ID NO: 7) (a₇)GHRDVARYLRAAAGGTRGSNHARIDAAEGPSDIPD;

(b) a functional equivalent of the fragment of (a) which is stillcapable of inducing an immune response against p16^(INK4a); or

(c) a combination of fragments of (a) and/or (b).

The term “functional equivalent” as used herein relates to, e.g.,variants or fragments of (a) which are still capable of inducing animmune response against p16^(INK4a), thus, are still useful as anefficient vaccine. The variants are characterized by amino aciddeletions, substitutions, and/or additions. Preferably, amino aciddifferences are due to one or more conservative amino acidsubstitutions. The term “conservative amino acid substitutions” involvesreplacement of the aliphatic or hydrophobic amino acids Ala, Val, Leuand Be; replacement of the hydroxyl residues Ser and Thr; replacement ofthe acidic residues Asp and Glu; replacement of the amide residues Asnand Gln, replacement of the basic residues Lys, Arg, and His;replacement of the aromatic residues Phe, Tyr, and Trp, and replacementof the small-sized amino acids Ala, Ser, Thr, Met, and Gly.

For the generation of peptides showing a particular degree of identity,e.g., genetic engineering can be used to introduce amino acid changes atspecific positions of a cloned DNA sequence to identify regions criticalfor peptide function. For example, site directed mutagenesis oralanine-scanning mutagenesis (introduction of single alanine mutationsat every residue in the molecule) can be used (Cunningham and Wells,1989). The resulting mutant molecules can then be tested forimmunogenicity using the assays of the examples.

Preferably, the variants are characterized by not more than 8 aa, morepreferably by not more than 6 aa and, even more preferably, by not morethan 4 aa substitutions, deletions and/or additions.

In the fragment of the original p16^(Ink4a)-fragment at least 5contiguous aa, preferably at least 10 contiguous aa, more preferably atleast contiguous 15 aa and even more preferably at least 20 contiguousaa of the particular amino acid sequence are left. Such fragment isstill capable of inducing an immune response against p16^(INK4a) and,thus, is still useful as an efficient vaccine.

The present invention also provides a nucleic acid encoding a fragmentof the invention or a vector containing such nucleic acid. The directinjection of genetic material into a living host causes a small amountof its cells to produce the introduced gene products. This inappropriategene expression within the host has important immunologicalconsequences, resulting in the specific immune activation of the hostagainst the gene delivered antigen. Direct injection of naked plasmidDNA induces strong immune responses to the antigen encoded by the genevaccine. Once the plasmid DNA construct is injected the host cells takeup the foreign DNA, expressing the viral gene and producing p16^(INK4a)inside the cell. This form of antigen presentation and processinginduces both MHC and class I and class II restricted cellular andhumoral immune responses. The DNA vaccines are composed of vectorsnormally containing two unites: the antigen expression unit composed ofpromoter/enhancer sequences, followed by antigen (FSP)-encoding andpolyadenylation sequences and the production unit composed of sequencesnecessary for vector amplification and selection. The construction ofvectors with vaccine inserts is accomplished using recombinant DNAtechnology and the person skilled in the art knows vectors that can beused for this approach. The efficiency of DNA immunization can beimproved by stabilising DNA against degradation, and increasing theefficiency of delivery of DNA into antigen presenting cells. This hasbeen demonstrated by coating biodegradable cationic microparticles (suchas poly(lactide-co-glycolide) formulated with cetyltrimethylammoniumbromide) with DNA. Such DNA-coated microparticles can be as effective atraising CTL as recombinant vaccinia viruses, especially when mixed withalum. Particles 300 nm in diameter appear to be most efficient foruptake by antigen presenting cells.

A variety of expression vectors, e.g., plasmids or viral vectors, may beutilised to contain and express nucleic acid sequences encoding afragment of the present invention.

A preferred viral vector is a poxvirus, adenovirus, retrovirus,herpesvirus or adeno-associated virus (AAV). Particularly preferredpoxviruses are a vaccinia virus, NYVAC, avipox virus, canarypox virus,ALVAC, ALVAC(2), fowlpox virus or TROVAC.

Recombinant alphavirus-based vectors have also been used to improve DNAvaccination efficiency. The gene encoding the fragment of the inventionis inserted into the alphavirus replicon, replacing structural genes butleaving non-structural replicase genes intact. The Sindbis virus andSemliki Forest virus have been used to build recombinant alphavirusreplicons. Unlike conventional DNA vaccinations, however, alphavirusvectors are only transiently expressed. Alphavirus replicons raise animmune response due to the high levels of protein expressed by thisvector, replicon-induced cytokine responses, or replicon-inducedapoptosis leading to enhanced antigen uptake by dendritic cells.

The present invention also provides a pharmaceutical compositioncontaining a fragment, nucleic acid sequence or vector of the presentinvention in an amount suitable for immunization of an individual and,preferably, one or more common auxiliary agents. Such a fragment,nucleic acid sequence or vector can be present as such or in combinationwith carriers. It is favourable for the carriers in the individual notto be immunogenic. Such carriers may be the individual's own proteins orforeign proteins or fragments thereof. Carriers, such as serum albumin,fibrinogen or transferrin or a fragment thereof are preferred. Thefragments contain epitopes which are recognized by cytotoxic T cells,e.g. CD8⁺ T cells or CD4 T cells, and may induce an immune response.Such epitopes of cell cycle regulatory proteins can be determined bymethods with which a person skilled in the art is familiar. It can alsobe advantageous that various fragments are simultaneously present. Forthe recombinant production of the above fragments, reference is made,e.g., to Sambrook et al., Molecular Cloning: A Laboratory Manual, 2^(nd)edition, Cold Spring Harbour Laboratory Press, Cold Spring Harbor N.Y.(1989).

The present invention also relates to the use of a fragment, nucleicacid sequence or vector of the present invention for the production of avaccine for preventing or treating a p16^(INK4a)-expressingpre-neoplasia, neoplasia or carcinoma (including an advanced carcinoma).

For example, these may be HPV-induced, p16^(INK4a)-expressing anogenitalcarcinomas, in particular cervical carcinoma, or head and neck cancerand non HPV-induced p16^(INK4a)-expressing tumors. Likewise benignmodifications such as papillomas, adenomas, hyperplasias or similarproliferations of epithelial, mesenchymal or hematopoieticproliferations are also to be counted there among.

The employed term “individual” comprises an individual of any kind andbeing able to fall ill with carcinomas. Examples of such individuals arehumans and animals as well as cells thereof.

The employed term “amount suitable for immunization of an individual”comprises any amount of a fragment of the invention, to which the aboveexplanations apply correspondingly and with which an individual can beimmunized. For example, the amount depends on whether immunization isintended as a prophylactic or therapeutic treatment. In addition, theindividual's age, sex and weight play a role for determining the amount.It is favourable to give the individual 100 μg to 1 g of a p16 fragmentby means of injection. The injection may be made at various sites of theindividual intramuscularly, subcutaneously, intradermally or in anyother form of application. It may also be favourable to carry out one ormore “booster injections” having about equal amount. In this case, itmay be particularly favourable to use different fragments of therespective cell cycle regulatory proteins for the individual injections.

The employed term “common auxiliary agents” comprises any auxiliaryagents suitable for a pharmaceutical composition to immunize anindividual. Such auxiliary agents are, e.g., immunization adjuvants,such as GM-CSF or Freund's adjuvant, buffered common salt solutions,water, emulsions, such as oil/water emulsions, wetting agents, sterilesolutions, etc.

By means of the present invention it is possible to immunizeindividuals, in particular humans and animals. Immunization takes placeby both induction of antibodies and stimulation of T cells. Thus, it ispossible to take prophylactic and therapeutic steps againstpre-neoplasias, neoplasias and carcinomas.

The invention is explained by the below examples.

Example 1 T Cell Reactivity Against p16^(INK4a) Peptides in Patientswith HPV-Associated Neoplasia

In order to evaluate whether and to what extent patients withHPV-associated tumors raise T cell responses against the stronglyoverexpressed p16^(INK4a), different methods were applied which allowfor a detailed characterization of immune responses against thep16^(INK4a) antigen. The finding of spontaneous immune responses againstp16^(INK4a) in cervical cancer patients proves the immunogenicity of theantigen in general and the particular p16INK4a fragments and providesthe rational for immunizing patients with p16^(INK4a) expressing tumorswith the p16INK4a fragments.

Peripheral blood mononuclear cells (PBMCs) from 13 women withp16^(INK4a)-expressing high grade cervical dysplasia (CIN2/3) wereincubated with p16^(INK4a) peptides (Table 1) to determine theproliferative capacity of the immune cells by applying a BrdU assay as aglobal measure for lymphoproliferative potential after challenge withthe p16^(INK4a) peptides. The BrdU-assay is a colorimetric immunoassayapplied for the quantification of cell proliferation based on themeasurement of the thymidine analogue 5-bromo-2′-deoxyuridineincorporation during DNA synthesis. PBMCs in IMDM medium supplementedwith 10% human serum were seeded in a 96-well-microtiter-plate (flatbottom) at a density of 150.000 cells/50 μl/well. Cells in each 4replicate wells were incubated in the presence of the seven p16INK4apeptides (Table 1), tetanus toxoid (20 ng/ml, Calbiochem, La Jolla,Calif.) and mitogen PHA-L (5 μg/ml, Roche, Mannheim, Germany) aspositive controls and no antigen as negative control for 6 days at 37°C., 5% CO2. On day 6, 10 μl/well of BrdU labelling solution (all BrdUassay reagents were used from the Cell Proliferation ELISA, BrdU(colorimetric) by Roche, Mannheim, Germany) diluted 1:100 in IMDMmedium+10% human serum were added to each well and incubated for anadditional period of 18 hours at 37° C., 5% CO2. On day 7, the plate wascentrifuged at 1200 rpm for 10 min and 50 μl of the supernatant wasremoved and transferred to a new V-bottom plate to be finally stored at−80° C. for cytokine analysis. The remaining cells were dried by using ahair dryer for about 15 minutes and 150 μl/well FixDenat solution wereadded to the cells and incubated for 30 minutes at room temperaturebefore removing FixDenat by flicking off and tapping carefully. 100μl/well anti-BrdU-POD working solution were added and incubated for 90minutes at room temperature, then removed and replaced by 100 μl TMBsubstrate, which was incubated for 30 minutes at room temperature. Theenzyme reaction was stopped by adding 25 μl/well 1N H₂SO₄ and opticaldensity (OD) was measured at 450 nm (reference wavelength 620 nm).Cut-off for positive reactions was set as three times standard deviationof ODs in the negative control wells without antigen. PBMCs from 3 outof the 13 tested women showed proliferation in response to thep16^(INK4a) peptides, indicating that incubation with the peptides hasactivated proliferative memory T cell responses. Overall the pattern ofresponse inducing peptides was heterogenic, indicating various T cellepitopes within the p16 antigen. One patient showed response to peptidesp16INK4a_(—)18-44, p16INK4a_(—)37-63, p16INK4a_(—)73-107 andp16INK4a_(—)123-156 one patient to peptides p16INK4a_(—)51-80 andp16INK4a_(—)98-128 and one patients to peptides p16INK4a_(—)1-25 andp16INK4a_(—)37-63 (FIG. 4).

In order to prove that particular p16 fragments are able to induceinterferon gamma secretion as one sign for a Th1 response, T cells from23 patients with invasive cervical cancer and high grade precancerouslesions (CIN2/3) with strong p16^(INK4a) overexpression were testedagainst the seven p16^(INK4a) peptides (Table 1) in interferon gammaELISpot assays. T cells were separated from heparinized blood usingFicoll centrifugation, plastic adherence and antibody coupled magneticbeads (CD11, CD16, CD19, CD36, CD56, Pan T cell isolation Kit, Milteny,Bergisch Gladbach, Germany). Dendritic cells were generated by culturingplastic adherent cells for 7 days with IL4 and GM-SCF (each 1000 U/ml)and used as antigen presenting cells in the ELIspot. Each 10⁵ T cellswere tested after a short (2 to 5 days) in vitro presensitization withthe respective peptide presented by 2×10⁴ dendritic cells.

When subtracting background (2 times the spots in the negative controlwell+2 standard deviations of reactivity against the respectivep16^(INK4a) peptide) in 7 cervical cancer patients T cells (CD4 or CD8)reacting against the p16INK4a_(—)37-63 peptide could be identified (FIG.2).

Example 2 In Vitro Priming of Healthy Donor T Cells with p16^(INK4a)Peptides

Seven long 25-35mer peptides covering the entire p16^(INK4a) amino acidsequence, each with a 7-13 amino acid overlap were tested to definep16^(INK4a) fragments which are able to induce interferon gammasecreting T cells from healthy donors in vitro. (Table 1).

TABLE 1 Seven overlapping p16^(INK4a) peptides usedin in vitro experiments SEQ ID No. peptide amino acid sequence 1p16INK4a_1-25 MEPAAGSSMEPSADWLATAAARGRV 2 p16INK4a_18-44TAAARGRVEEVRALLEAGALPNAPNSY 3 p16INK4a_37-63 LPNAPNSYGRRPIQVMMMGSARVAELL4 p16INK4a_51-80 VMMMGSARVAELLLLHGAEPNCADPATLTR 5 p16INK4a_73-104ADPATLTRPVHDAAREGFLDTLVVLHRAGARL 6 p16INK4a_98-128HRAGARLDVRDAWGRLPVDLAEELGHRDVAR 7 p16INK4a_GHRDVARYLRAAAGGTRGSNHARIDAAEGPSDI 123-156 PD

In order to show that p16^(INK4a) fragments can stimulate healthy donorT cells in vitro to secrete interferon gamma and to identify the mostimmunogenic p16^(INK4a) derived epitopes, it was investigated whether Tcells isolated from peripheral blood of healthy donors can be stimulatedin vitro with these p16^(INK4a) peptides. If the p16^(INK4a) peptidesare able to induce a specific T cell response in cell cultureexperiments, the T cells secrete cytokines when challenged with therespective p16^(INK4a) peptide in so called ELISpot experiments. InELISpot assays the cytokines (interferon gamma) can be detected byspecific antibodies with a subsequent colour reaction.

Peripheral blood mononuclear cells (PBMC) were isolated from heparinizedblood (100 ml) of one healthy donor by a Ficoll Plaque gradient densitycentrifugation. 5 to 10×10⁷ PBMC were separated into monocytes and Tcells by plastic adherence and antibody coupled magnetic beads (CD11,CD16, CD19, CD36, CD56, Pan T cell isolation Kit, Milteny, BergischGladbach, Germany). The monocytes were cultured over 7 days with GM-CSFand IL-4 (each 1000 U/ml) to generate antigen presenting dendriticcells.

2×10⁷ T cells were incubated with 2×10⁶ dendritic cells that were priorpulsed with the p16^(INK4a) peptides (10 μg/ml) for 4 hours to achievepresentation of the antigens. For each of the 7 p16^(INK4a) peptides aseparate stimulation approach was processed.

The T cells were re-stimulated with p16^(INK4a) peptide pulsed dendriticcells and treated with IL-2 and IL-7 (10 U/ml) every 7 days over a 5week period.

The p16^(INK4a) peptide specific T cell response was measured ininterferon gamma ELISpot assays before the stimulations (day 0) andafter the last stimulation (day 35). For ELISpot assays, 96 wellnitrocellulose plates (MAHA N4550 Millipore) were coated withanti-interferon gamma antibody 1-D1K (Mabtech, Nacka Strand, Sweden) ata concentration of 0.75 ug/well. Each 10⁵ T cells were tested with therespective peptide presented by 2×10⁴ dendritic cells. After 12 hoursincubation at 37° C., detection of secreted interferon gamma wasachieved by detection with a biotinylated secondary anti-interferongamma antibody 7BG-1 (Mabtech), streptavidin-alkaline phosphataseconjugate and BCIP/NBT substrate solution (Sigma Aldrich, St. Louis,USA).

While no reactivity was detectable on day 0 against any of the sevenp16^(INK4a) peptides but only against a virus peptide mix (CEF=CMV & EBV& flu) used as positive control, on day 35 the T cells stimulated withpeptides p16INK4a_(—)37-63, p16INK4a_(—)51-80 and p16INK4a_(—)73-104showed an increased interferon gamma secretion in the ELISpot whentested against target cells pulsed with the respective p16^(INK4a)peptides but not when tested against cells pulsed with the remainingp16^(INK4a) peptides (FIG. 1).

Example 3 Lysis of Cervical Cancer Cell Lines by p16^(INK4a) Reactive TCells

The ability of activated T cells to lyze p16^(INK4a) expressing cervicalcancer cells was tested by chromium release assays using differentcervical cancer cell lines as well as HLA matched B cells loaded withp16^(INK4a) peptides as targets. 10×6 target cells (peptide loadedHLA-matched B cells, B cells without peptide) were incubated for 1 hourwith ⁵¹Cr (100 μCi) and afterwards with different ratios of T cells fromone representative cervical cancer patient that reacted against targetcells loaded with the peptide p16INK4a_(—)37-63 in ELIspot assays.Specific lysis of target cells by the T cells can be measured bydetection of released radioactivity.

It could be shown that the CD8+ T cells of a cervical cancer patientthat reacted against target cells loaded with the peptidep16INK4a_(—)37-63 in ELIspot assays were able to lyse HLA-matched andp16INK4a_(—)37-63-peptide loaded B cells as well as the cervical cancercell lines HeLa and Caski (both HPV and p16^(INK4a) positive), while nolysis could be detected with the same HLA-matched B cells without thep16INK4a_(—)37-63 peptide and other HPV- and p16^(INK4a) negative celllines (FIG. 3). These results demonstrate the cytotoxic activity ofp16^(INK4a)-peptide specific T cells and demonstrate thatp16^(INK4a)-epitopes are presented on cervical cancer cells andrecognized by patient's p16^(INK4a) reactive T cell clones.

Example 4 Humoral Immune Response Against p16^(INK4a)

The analysis of over 900 sera further demonstrated that a fraction ofindividuals develops antibodies that specifically bind to p16^(INK4a)derived epitopes (FIG. 4) (Reuschenbach et al., 2008), which shows theability of p16^(INK4a) to induce humoral immune responses.

Further is was demonstrated, that also against the peptidep16INK4a_(—)37-63 which appeared to induce the strongest interferongamma secretion by T cells in vitro, antibodies can be detected in sera.A total of 374 sera from patients with cervical cancer, small cell lungcancer, head and neck cancer and healthy individuals were tested inpeptide ELISAs. The peptide (20 ug/ml) was coated to 96 well microtiterplates (Maxisorp, Nunc, Roskilde, Denmark) over night at 4° C.,non-specific bindings sites were blocked with 0.5% casein and the serawere tested 1:100 for antibodies against the peptide. Bound serumantibodies were detected by HRP-conjugated anti-human IgG antibody(Jackson Immuno, West Grove, USA and TMB substrate (Sigma Aldrich, St.Louis, USA). At a cut off of a background normalized optical density of0.03, 15% (56/374) of the tested sera had antibodies againstp16INK4a_(—)37-63.

Taken together, these observations demonstrate that patients sufferingfrom HR-HPV-induced dysplasia or neoplasia are capable to develop ananti-p16^(INK4a)-immune response. If this immune response would becapable to eliminate HR-HPV transformed cells, the p16^(INK4a) antigenmight represent a very attractive candidate for forthcoming vaccines toprevent and/or treat p16INK4a-expressing (pre)-neoplasia, particularlyHR-HPV-induced tumors.

LIST OF REFERENCES

-   Beauséjour, C. M., Krtolica, A., Galimi, F., Narita, M., Lowe, S.    W., Yaswen, P., and Campisi, J. (2003). Reversal of human cellular    senescence: roles of the p53 and p16 pathways. EMBO J 22, 4212.-   Cunningham and Wells, Science 244 (1989), 1081-1085.-   Finn, O. J. (2008). Cancer immunology. N. Engl. J. Med. 358,    2704-2715.-   Hildesheim, A., Herrero, R., Wacholder, S., Rodriguez, A. C.,    Solomon, D., Bratti, M. C., Schiller, J. T., Gonzalez, P., Dubin,    G., Porras, C., Jimenez, S. E., and Lowy, D. R. (2007). Effect of    human papillomavirus 16/18 L1 viruslike particle vaccine among young    women with preexisting infection: a randomized trial. JAMA 298,    743-753.-   Jäger, E., Jäger, D., and Knuth, A. (2003). Antigen-specific    immunotherapy and cancer vaccines. Int J Cancer 106, 817-20.-   Klaes, R., Friedrich, T., Spitkovsky, D., Ridder, R., Rudy, W.,    Petry, U., Dallenbach-Hellweg, G., Schmidt, D., and Knebel    Doeberitz, M. (2001). Overexpression of p16(INK4A) as a specific    marker for dysplastic and neoplastic epithelial cells of the cervix    uteri. Int J Cancer 92, 276.-   Parkin, D. M. and Bray, F. (2006). Chapter 2: The burden of    HPV-related cancers. Vaccine 24 Suppl 3, S11-S25.-   Rescigno, M., Avogadri, F., and Curigliano, G. (2007). Challenges    and prospects of immunotherapy as cancer treatment. Biochim.    Biophys. Acta 1776, 108-123.-   Reuschenbach, M., Waterboer, T., Wallin, K. L., Einenkel, J.,    Dillner, J., Hamsikova, E., Eschenbach, D., Zimmer, H., Heilig, B.,    Kopitz, J., Pawlita, M., Doeberitz, M. K., and Wentzensen, N.    (2008). Characterization of humoral immune responses against p16,    p53, HPV16 E6 and HPV16 E7 in patients with HPV-associated cancers.    Int. J. Cancer 123, 2626-2631.-   Sano, T., Oyama, T., Kashiwabara, K., Fukuda, T., and Nakajima, T.    (1998). Expression status of p16 protein is associated with human    papillomavirus oncogenic potential in cervical and genital lesions.    Am. J. Pathol. 153, 1741-1748.-   Schadlich, L., Senger, T., Gerlach, B., Mucke, N., Klein, C.,    Bravo, I. G., Muller, M., and Gissmann, L. (2009). Analysis of    modified human papillomavirus type 16 L1 capsomeres: the ability to    assemble into larger particles correlates with higher    immunogenicity. J. Virol. 83, 7690-7705.-   Ishikawa M, Fujii T, Saito M, Nindl I, Ono A, Kubushiro K et al.    Overexpression of p16 INK4a as an indicator for human papillomavirus    oncogenic activity in cervical squamous neoplasia. Int J Gynecol    Cancer 2006; 16(1):347-353.-   Samama B, Lipsker D, Boehm N. p16 expression in relation to human    papillomavirus in anogenital lesions. Hum Pathol 2006;    37(5):513-519.-   Missaoui N, Hmissa S, Frappart L, Trabelsi A, Ben Abdelkader A,    Traore C et al. p16INK4A overexpression and HPV infection in uterine    cervix adenocarcinoma. Virchows Arch 2006; 448(5):597-603.-   Santos M, Landolfi S, Olivella A, Lloveras B, Klaustermeier J,    Suarez H et al. p16 overexpression identifies HPV-positive vulvar    squamous cell carcinomas. Am J Surg Pathol 2006; 30(11):1347-1356.-   Roma A A, Goldblum J R, Fazio V, Yang B. Expression of 14-3-3sigma,    p16 and p53 proteins in anal squamous intraepithelial neoplasm and    squamous cell carcinoma. Int J Clin Exp Pathol 2008; 1(5):419-425.-   Hafkamp H C, Speel E J M, Haesevoets A, Bot F J, Dinjens W N M,    Ramaekers F C S et al. A subset of head and neck squamous cell    carcinomas exhibits integration of HPV 16/18 DNA and overexpression    of p16INK4A and p53 in the absence of mutations in p53 exons 5-8.    Int J Cancer 2003; 107(3):394.-   Nindl I, Meyer T, Schmook T, Ulrich C, Ridder R, Audring H et al.    Human papillomavirus and overexpression of P16INK4a in nonmelanoma    skin cancer. Dermatol Surg 2004; 30(3):409-414.-   Busch C, Geisler J, Knappskog S, Lillehaug J R, Lønning PE.    Alterations in the p53 pathway and p16INK4a expression predict    overall survival in metastatic melanoma patients treated with    dacarbazine. J Invest Dermatol. 2010 October; 130(10):2514-6.-   Giordano G, Azzoni C, D'Adda T, Rocco A, Gnetti L, Froio E et al.    Human papilloma virus (HPV) status, p16INK4a, and p53 overexpression    in epithelial malignant and borderline ovarian neoplasms. Pathol Res    Pract 2008; 204(3):163-174.-   Giordano G, Azzoni C, D'Adda T, Merisio C. P16(INK4a) overexpression    independent of Human Papilloma Virus (HPV) infection in rare    subtypes of endometrial carcinomas. Pathol Res Pract. 2007;    203(7):533-8.-   Di Vinci A, Perdelli L, Banelli B, Salvi S, Casciano I, Gelvi I,    Allemanni G, Margallo E, Gatteschi B, Romani M. p16(INK4a) promoter    methylation and protein expression in breast fibroadenoma and    carcinoma. Int J Cancer. 2005 Apr. 10; 114(3):414-21.-   Buza N, Cohen P J, Pei Hui, Parkash V. Inverse p16 and p63    expression in small cell carcinoma and high-grade urothelial cell    carcinoma of the urinary bladder. Int J Surg Pathol. 2010 April; 18    (2):94-102.-   Esposito V, Baldi A, Tonini G, Vincenzi B, Santini M, Ambrogi V,    Mineo T C, Persichetti P, Liuzzi G, Montesarchio V, Wolner E, Baldi    F, Groeger A M. Analysis of cell cycle regulator proteins in    non-small cell lung cancer. J Clin Pathol. 2004 January;    57(1):58-63.-   Ikuerowo S O, Kuczyk M A, von Wasielewski R, shitu OB, Jonas U,    Machtens S, Serth J. p16INK4a expression and clinicopathologic    parameters in renal cell carcinoma. Eur Urol. 2007 March;    51(3):732-7; discussion 738. Epub 2006 Aug. 23.-   Leversha M A, Fielding P, Watson S, Gosney J R, Field J K.    Expression of p53, pRB, and p16 in lung tumours: a validation study    on tissue microarrays. J Pathol. 2003 August; 200(5):610-9.-   Ding G C, Ren J L, Chang F B, Li J L, Yuan L, Song X, Zhou S L, Guo    T, Fan Z M, Zeng Y, Wang L D. Human papillomavirus DNA and    P16(INK4A) expression in concurrent esophageal and gastric cardia    cancers. World J Gastroenterol. 2010 Dec. 14; 16(46):5901-6.-   Kim B N, Yamamoto H, Ikeda K, Damdinsuren B, Sugita Y, Ngan C Y,    Fujie Y, Ogawa M, Hata T, Ikeda M, Ohue M, Sekimoto M, Monden T,    Matsuura N, Monden M. Methylation and expression of p16INK4 tumor    suppressor gene in primary colorectal cancer tissues. Int J Oncol.    2005 May; 26(5):1217-26.-   Okamoto A, Demetrick D J, Spillare E A, Hagiwara K, Hussain S P,    Bennett W P, Forrester K, Gerwin B, Greenblatt M S, Serrano M.    p16INK4 mutations and altered expression in human tumors and cell    lines. Cold Spring Harb Symp Quant Biol 1994; 59:49-57.

What is claimed is:
 1. A vector comprising a nucleic acid encoding afragment of the cyclin-dependent kinase inhibitor p16^(INK4) capable ofinducing an immune response against p16^(INK4a), wherein said nucleicacid encodes: (a) any one of the following amino acid sequences:(SEQ ID NO: 1) (a₁) MEPAAGSSMEPSADWLATAAARGRV; (SEQ ID NO: 2) (a₂)TAAARGRVEEVRALLEAGALPNAPNSY; (SEQ ID NO: 3) (a₃)LPNAPNSYGRRPIQVMMMGSARVAELL; (SEQ ID NO: 4) (a₄)VMMMGSARVAELLLLHGAEPNCADPATLTR; (SEQ ID NO: 5) (a₅)ADPATLTRPVHDAAREGFLDTLVVLHRAGARL; (SEQ ID NO: 6) (a₆)HRAGARLDVRDAWGRLPVDLAEELGHRDVAR; (SEQ ID NO: 7) (a₇)GHRDVARYLRAAAGGTRGSNHARIDAAEGPSDIPD;

(b) a functional equivalent of the fragment of (a) which is stillcapable of inducing an immune response against p16^(INK4a); or (c) acombination of fragments of (a) and/or (b).
 2. The vector of claim 1,wherein said vector is a plasmid or viral vector.
 3. The viral vector ofclaim 2, wherein said viral vector is a poxvirus, adenovirus,retrovirus, herpesvirus, alphavirus-based vector or adeno-associatedvirus (AAV).
 4. The viral vector of claim 3, wherein said poxvirus is avaccinia virus, NYVAC, avipox virus, canarypox virus, ALVAC, ALVAC(2),fowlpox virus or TROVAC.
 5. A pharmaceutical composition containing thevector of claim
 1. 6. The pharmaceutical composition of claim 5, whereinthe composition is a vaccine.
 7. A method for preventing or treating ap16-expressing pre-neoplasia, neoplasia or tumor, comprisingadministering the vector of claim 1 or the amino acid sequence encodedby the nucleic acid of the vector of claim
 1. 8. The method according toclaim 7, wherein said tumor is an advanced carcinoma.
 9. The methodaccording to claim 7, wherein said tumor is an advanced sarcoma.
 10. Themethod according to claim 7, wherein said tumor is an anogenital tumoror head and neck tumor.
 11. The method according to claim 7, whereinsaid tumor is a tumor of the esophago-gastro-intestinal tract, a tumorof the hepato-pancreato-biliary tract, a tumor of the respiratory tract,or a skin tumor.
 12. The method according to claim 7, wherein said tumoris a tumor of the kidney and urinary tract, mamma, ovary, endometrium,or prostate.
 13. The method according to claim 7, wherein said tumor isa tumor of the nervous system.
 14. The method according to claim 7,wherein said tumor is an HPV-induced tumor.
 15. The method according toclaim 10, wherein said anogenital carcinoma is a cervical carcinoma.